Effect of 5-methylcytosine on the stability of triple-stranded DNA--a thermodynamic study.

We have previously shown that the pyrimidine oligonucleotide 5'CTTCCTCCTCT (Y11) recognizes the double-helical stem of hairpin 5'GAAGGAGGAGA-T4-TCTCCTCCTTC (h26) by triple-helix formation (1). In this paper, we report the effect on triplex formation of substituting the cytosine residues of Y11 with 5-methylcytosines (5meY11). In addition, we have studied the thermodynamics of the interaction between h26 and 5meY11. The results can be summarised as follows: (i) gel electrophoresis shows that at T = 5 degrees C and pH 5, both Y11 and 5meY11 form DNA triple helices with h26, whereas at pH 6.8 only the methylated strand binds to h26; (ii) pH-stability curves of the DNA triplexes formed from h26 + Y11 and h26 + 5meY11 show that Y11 and 5meY11 are semi-protonated at pH 5.7 and 6.7, respectively. Thus, it is concluded that cytosine methylation expands the pH range compatible with triplex formation by one pH unit; (iii) as the unmethylated triplex (h26:Y11), the methylated one (h26:5meY11) denatures in a biphasic manner, in which the low temperature transition results from the dissociation of 5meY11 from h26. The Tm of the triplex to h26 plus 5meY11 transition is strongly enhanced (about 10 degrees C) by cytosine methylation. A van 't Hoff analysis of denaturation curves is presented; (iv) DSC experiments show that triplex formation between 5meY11 and h26 is characterized by delta H = -237 +/- 25 kJ/mol and delta S = -758 +/- 75 J/Kmol, corresponding to an average delta H of -21 kJ/mol and delta S of -69 J/Kmol per Hoogsteen base pair; (v) the thermodynamic analysis indicates that the extra stability imparted to the triplex by methylcytosine is entropic in origin.     

Detection and kinetic studies of triplex formation by oligodeoxynucleotides using real-time biomolecular interaction analysis (BIA).

Real-time biomolecular interaction analysis (BIA) has been applied to triplex formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand. Sequence-specific triplex formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surface was able to capture a DNA duplex by triplex recognition. The presence of spermine increases the rate of association between the third strand and immobilised duplex, but at elevated spermine concentrations non-specific association is observed. A preliminary kinetic analysis of triplex formation at pH 5.2 by an 11mer third strand containing thymine, cytosine and uracil is reported. Values for the association and dissociation rate constants were determined to be (1.9 +/- 0.2) x 10(3) M-1 s-1 and (8.1 +/- 1.9) x 10(-5) s-1, respectively.     

Influence of sequence-dependent cytosine protonation and methylation on DNA triplex stability

To investigate cytosine protonation and its influence on the sequence-dependent thermal stability of DNA triplexes in detail, we have employed homo- and heteronuclear NMR experiments on specifically (15)N-labeled oligodeoxynucleotides that were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. These experiments reveal that cytosines in central positions of the triplex are significantly protonated even at neutral pH. However, semiprotonation points for individual cytosine bases as determined from pH-dependent measurements show considerable differences depending on their position. Thus, protonation is disfavored for adjacent cytosines or for cytosines at the triplex termini, resulting in a smaller contribution to the overall free energy of the triple helical system. In contrast, protonation of the base upon substitution of 5-methylcytosine for cytosine in the triplex third strand is only affected to a minor extent, and triplex stabilization by the methyl substituent is shown to primarily arise from stacking energies and/or hydrophobic effects.     

Physico-chemical studies of a DNA triplex containing a new ferrocenemethyl-thymidine residue in the third strand

The stability of a 16-mer DNA triple helix containing a 3-N(ferrocenemethyl)-thymidine residue in the third strand has been investigated in comparison with the unmodified triplex of the same sequence. A complete physico-chemical characterization of the two triple helices on changing the pH by means of calorimetry, circular dichroism and molecular modeling is therefore reported. The thermodynamic parameters were obtained in the pH range 5.5-7.2 by differential scanning calorimetry (DSC). For both triplexes the T(m) and Delta H degrees (T(m)) values increase on decreasing the pH. In the pH range 7.2-6.0 the triplex containing the ferrocenemethyl nucleoside is less stable than the unmodified one, whereas the modified triplex becomes more stable at pH 5.5. Such difference in stability at each pH value is overwhelmingly enthalpic in origin. CD spectra show conformational changes on decreasing the pH for both the triplexes. By spectroscopic pH titration the apparent pK(a) values of the cytosines in the two triplexes could be estimated, with the cytosines in the TFO containing the ferrocenemethyl residue having lower apparent pK(a) values. These results are consistent with the calorimetric data, showing a decrease of the thermodynamic parameters in the pH range 7.2-6.0 and an increase at pH 5.5 for the ferrocenylated triplex with respect to the unmodified one. The thermodynamic and spectroscopic data are also discussed in relation to molecular models.     

2'-O,4'-C-ethylene bridged nucleic acid modification enhances pyrimidine motif triplex-forming ability under physiological condition

Since pyrimidine motif triplex DNA is unstable at physiological neutral pH, triplex stabilization at physiological neutral pH is important for improvement of its potential to be applied to various methods in vivo, such as repression of gene expression, mapping of genomic DNA and gene-targeted mutagenesis. For this purpose, we studied the thermodynamic and kinetic effects of a chemical modification, 2'-O,4'-C-ethylene bridged nucleic acid (ENA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological neutral pH. Thermodynamic investigations indicated that the modification achieved more than 10-fold increase in the binding constant of the triplex formation. The increased number of the modification in TFO enhanced the increased magnitude of the binding constant. On the basis of the obtained thermodynamic parameters, we suggested that the remarkably increased binding constant by the modification may result from the increased stiffness of TFO in the unbound state. Kinetic studies showed that the considerably decreased dissociation rate constant resulted in the observed increased binding constant by the modification. We conclude that ENA modification of TFO could be a useful chemical modification to promote the triplex formation under physiological neutral condition, and may advance various triplex formation-based methods in vivo.     

三链DNA稳定性的影响因素

介绍了碱基组成、碱基修饰或替代、DNA骨架的修饰、DNA配体的结合及反应体系中的盐离子和pH值等因素对三链DNA稳定性的影响。对三链DNA稳定性研究中应注意的几个问题也作了讨论。     

pH and cation effects on the properties of parallel pyrimidine motif DNA triplexes

The effects of cytosine protonation and various cations on the properties of parallel pyrimidine motif DNA triplexes were intensively investigated and characterized by several different techniques, such as circular dichroism (CD) conformation, ultraviolet (UV) melting, differential scanning calorimetry (DSC) thermal denaturation, and surface plasmon resonance (SPR) real-time dynamics. The comparative CD spectra of the triplex and the corresponding homoduplexes showed that the negative peak at approximately 218 nm would be the eigenpeak of the Hoogsteen paired strand, and moreover, the formation pathway of a triplex was significantly pH-dependent and fell into three groups: under acidic conditions, the triplex is formed by a one-step docking, under near physiological conditions, the Watson-Crick duplex is first structured and then accepts the Hoogsteen third strand into its major groove, and under basic conditions, the triplex is not formed. The pH-dependent thermodynamics of the global triplex, the Watson-Crick antiparallel duplex, and the Crick-Hoogsteen parallel duplex were comparatively discussed for the first time. These data revealed that the thermodynamic stabilities of the Watson-Crick-Hoogsteen triplex and the Crick-Hoogsteen duplex would be strongly dependent on cytosine protonation, but a low-pH environment somewhat destabilized the Watson-Crick duplex. The binding energy of triplex formation would be different from the unfolding energy of triplex melting under acidic conditions due to the disparity in the pathway between the formation and unfolding of a triplex. Real-time dynamic measurements showed that the association and dissociation rate constants of a duplex-to-triplex formation are (1.98 +/- 0.24) x 10(3) M(-1) s(-1) and (4.09 +/- 0.96) x 10(-4) s(-1) at 20 degrees C and pH 6.0, respectively. The formation energy of the duplex-to-triplex transition derived from SPR measurements was in agreement with the unfolding energy of the free Hoogsteen paired duplex derived from UV measurements. The calorimetric enthalpies of the triplex-to-duplex-to-single transition were 39.3 and 75.3 kcal/mol under near physiological conditions (pH 7.0), respectively, which were underestimated relative to the van't Hoff enthalpies. In addition, the effects of various cations, ionic strength, mixed-valent cations, and the position of the C(+)xG.C triplets on the thermodynamics of the triplexes were addressed under near physiological conditions. The interaction of metal ions with the triplexes clearly depended on the type and ionic strength of the cations, and the efficiency with which the cations stabilized the global triplex was in the order Mg(2+) > Mn(2+) > Ca(2+) > Ba(2+) > Na(+). These observations would be useful for the design of triplex-forming oligonucleotides for antigene drugs and therapeutic purposes.     

Poly(L-lysine)-graft-dextran copolymer promotes pyrimidine motif triplex DNA formation at physiological pH. Thermodynamic and kinetic studies

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.     

2'-O,4'-C-methylene bridged nucleic acid modification promotes pyrimidine motif triplex DNA formation at physiological pH: thermodynamic and kinetic studies

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in an artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is, therefore, crucial in improving its therapeutic potential. To this end, we have investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO), on pyrimidine motif triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA modification of TFO increased the binding constant of the pyrimidine motif triplex formation at neutral pH by approximately 20 times. The number and position of the 2',4'-BNA modification introduced into the TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity itself of the 2',4'-BNA-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant at neutral pH. Kinetic data demonstrated that the observed increase in the binding constant at neutral pH by the 2',4'-BNA modification of TFO resulted from the considerable decrease in the dissociation rate constant. Our results certainly support the idea that the 2',4'-BNA modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.     

Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2'-O and 4'-C of the sugar moiety were bridged with the methylene chain and 3'-O was replaced by 3'-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo.     

High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA–dsDNA triplexes—mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na⁺). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally located mismatches was more than 150-fold. Together the data support the use of homopyrimidine PNAs as efficient and sequence selective tools in triplex targeting strategies under physiological relevant conditions.     

Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH

The sequence-specific recognition of duplex DNA by unmodified parallel triplex-forming oligonucleotides is restricted to low pH conditions due to a necessity for cytosine protonation in the third strand. This has severely restricted their use as gene-targeting agents, as well as for the detection and/or functionalisation of synthetic or genomic DNA. Here I report that the nucleobase 6-amino-5-nitropyridin-2-one (Z) finally overcomes this constraint by acting as an uncharged mimic of protonated cytosine. Synthetic TFOs containing the nucleobase enabled stable and selective triplex formation at oligopurine-oligopyrimidine sequences containing multiple isolated or contiguous GC base pairs at neutral pH and above. Moreover, I demonstrate a universal strategy for the enzymatic assembly of Z-containing TFOs using its commercially available deoxyribonucleotide triphosphate. These findings seek to improve not only the recognition properties of TFOs but also the cost and/or expertise associated with their chemical syntheses.     

Thermodynamic and kinetic effects of N3'-->P5' phosphoramidate modification on pyrimidine motif triplex DNA formation

I have investigated the thermodynamic and kinetic effects of N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation between a 23-bp target duplex and a 15-mer TFO using electrophoretic mobility shift assay, UV melting, isothermal titration calorimetry, and interaction analysis system. The thermodynamic and kinetic analyses have clearly indicated that the PN modification of TFO not only significantly increased the thermal stability of the pyrimidine motif triplex at neutral pH but also increased the binding constant of the pyrimidine motif triplex formation at room temperature and neutral pH by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters has suggested that the more rigidity of the PN TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant of the pyrimidine motif triplex formation at neutral pH. Kinetic data have also demonstrated that the observed PN modification-mediated promotion of pyrimidine motif triplex formation at neutral pH resulted from the considerable decrease in the dissociation rate constant rather than the increase in the association rate constant. This information will present an effective approach for designing chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, which may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.     

Comparative circular dichroism and fluorescence studies of oligodeoxyribonucleotide and oligodeoxyribonucleoside methylphosphonate pyrimidine strands in duplex and triplex formation

An analogue of the homopyrimidine oligodeoxyribonucleotide d(CT)8 has been synthesized. This analogue, d(CT)8 contains nonionic methylphosphonate internucleoside linkages. The pH-dependent conformational transitions of d(CT)8 have been studied and its ability to form duplexes and triplexes with the normal homopurine oligonucleotide d(AG)8 has also been investigated as a function of pH. Circular dichroism spectroscopy and ethidium bromide fluorescence enhancement have been used to monitor pH-dependent conformational transitions driven by the protonation of cytosine residues, and the different behavior of d(CT)8 and d(CT)8 has been compared. It was possible to form self-associated complexes by using either d(CT)8 or d(CT)8, and both compounds combined with d(AG)8 to form duplex or triplex DNA. At neutral pH, the CD spectrum of d(AG)8.d(CT)8 duplex was quite different from the CD spectrum of d(AG)8.d(CT)8 duplex, reflecting most likely a difference in conformation. The duplex to triplex transition characteristic of this DNA sequence occurred at a lower pH when d(CT)8 was substituted for d(CT)8; however, at pH 4.2, triplex containing d(CT)8 was similar in conformation to triplex containing d(CT)8. Several of these observations can be related to the alterations in electrostatic and steric interactions that occur when the negatively charged phosphodiester backbone of d(CT)8 is replaced with a nonionic methylphosphonate backbone.     

Sequence-dependent stability of intramolecular DNA triple helices

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.     

Hydrolysis of N3-methyl-2'-deoxycytidine: model compound for reactivity of protonated cytosine residues in DNA

Protonation of cytosine residues at physiological pH may occur in DNA as a consequence of both alkylation and aberrant base-pair formation. When cytosine derivatives are protonated, they undergo hydrolysis reactions at elevated rates and can either deaminate to form the corresponding uracil derivatives or depyrimidinate generating abasic sites. The kinetic parameters for reaction of protonated cytosine are derived by studying the hydrolysis of N3-methyl-2'-deoxycytidine (m3dC), a cytosine analogue which is predominantly protonated at physiological pH. Both deamination and depyrimidimation reaction rates are shown to be linearly dependent upon the fraction of protonated molecules. We present here thermodynamic parameters which allow determination of hydrolysis rates of m3dC as functions of pH and temperature. Protonation of cytosine residues in DNA, as induced by aberrant base-pair formation or base modification, may accelerate the rate of both deamination and depyrimidation up to several thousand-fold under physiological conditions.     

A molecular beacon strategy for real-time monitoring of triplex DNA formation kinetics

We used a molecular beacon (MB) containing a 15-mer triplex-forming oligonucleotide (TFO) to probe in real-time the kinetics of triplex DNA formation in the left side of the TCl tract (502-516) of the c-src proto-oncogene in vitro. The metal ions Na+, K+, and Mg2+ stabilized triplex DNA at this site. The pseudo-first-order rate constant (kpsi) and the second-order association rate constant (k1) for the binding of the MB to the target duplex in 10 mM sodium phosphate buffer, pH 7.3, increased from 3.2 +/- 0.9 to 15 +/- 2.8 x 10(-3) s(-1) and 6.4 +/- 1.8 to 30 +/- 5.6 x 102 M(-1) s(-1), respectively, on increasing the MgCl2 concentration from 1 to 2.5 mM. Similar values were obtained for the triplex DNA stabilized by NaCl (100-250 mM). Surprisingly, the values were around 2 times higher in the presence of KCl. The AG of triplex formation in the presence of 1 mM MgCl2, 150 mM NaCl, and 150 mM KCl were -7.8 +/- 0.3, -8.2 +/- 0.3 and -8.7 +/- 0.7 kcal/mol respectively, despite significant differences in the values of deltaH and deltaS, suggesting enthalpy-entropy compensation in the stabilization of the triplex DNA by these metal ions. These results show the utility of MBs ih probing triplex DNA formation and in evaluating kinetic and thermodynamic parameters important for the design and development of TFOs as triplex DNA-based therapeutic agents.     

Quantitative analysis of the ion-dependent folding stability of DNA triplexes

A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion dependence of the thermodynamic stability for DNA triplexes. We focus on two experimentally studied systems: a 24-base DNA triplex and a pair of interacting 14-base triplexes. Our theoretical calculations for the number of bound ions indicate that the TBI model provides improved predictions for the number of bound ions than the classical Poisson-Boltzmann (PB) equation. The improvement is more significant for a triplex, which has a higher charge density than a duplex. This is possibly due to the higher ion concentration around the triplex and hence a stronger ion correlation effect for a triplex. In addition, our analysis for the free energy landscape for a pair of 14-mer triplexes immersed in an ionic solution shows that divalent ions could induce an attractive force between the triplexes. Furthermore, we investigate how the protonated cytosines in the triplexes affect the stability of the triplex helices.     

Sequence-Dependent Stability of Intramolecular DNA Triple Helices

Abstract

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.     

A-tract DNA disfavours triplex formation.

Optimisation of DNA triplex stability is of fundamental importance in the anti-gene strategy. In the present work, thermal denaturation studies by UV-spectrophotometry and structural and dynamical characterizations by NMR spectroscopy have been used systematically to investigate the effects on triplex stability of isolated insertions of different base triplets into an otherwise homogeneous 15-mer dT x dA-dT oligo-triplex. It is found that insertion of a single central C(+) x G-C or T x D-T triplet (D=2,6-diaminopurine) leads to a pronounced stabilization (up to 20 deg. C if the cytosine base is C5 methylated) at acidic as well as neutral pH. To a smaller degree, this is the case also for a C(+) x I-C triplet insertion.Using imino proton exchange measurements, it is shown that insertion of a DT base-pair in the underlying duplex perturbs the intrinsic A-tract structure in the same way as has been shown for a GC insert. We propose that the intrinsic properties of A-tract duplex DNA (e. g. high propeller twist and rigidity) are unfavourable for triplex formation and that GC- or DT-inserts stabilize the triplex by interfering with the A-tract features of the underlying duplex. The C(+) x I-C triplet without the N2 amino group in the minor groove is readily accommodated within the typical, highly propeller-twisted A-tract structure. This might be related to its smaller effect on the stability of the corresponding triplex.These results may be valuable for understanding DNA triplex formation in vivo as well as for the design of efficient triplex-forming oligonucleotides and in choosing suitable target sequences in the anti-gene strategy.